Method and apparatus for three label microarrays

ABSTRACT

A method of and apparatus for directly visualizing printed microarrays are disclosed. In one embodiment, the method comprises the steps of (a) generating labeled probes labeled with a first label, (b) constructing a microarray with the labeled probes, wherein the microarray comprises a plurality of probe spots, and (c) examining the microarray to determine the amount of probe present at each probe spot.

CROSS-REFERENCE TO RELATED APPLICATION

The application claims priority to U.S. Ser. No. 60/314,005, filed Aug.21, 2001 and incorporated by reference.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENTBACKGROUND OF THE INVENTION

The cDNA microarray platform has great potential to generate newinsights into human disease (Dhanasekaran, et al., 2001; Garber, et al.,2001; Hedenfalk, et al., 2001; Hegde, et al., 2001; Schena, et al.,1995; Schena, et al., 1996; Sorlie, et al., 2001). The use of cDNAmicroarrays begins with construction of the array, where typically,hundreds to thousands of cDNA probes are amplified by PCR, purified, andprinted onto coated glass slides (typically poly-L-lysine or aminosaline). In a typical experiment, slides are fixed, blocked, and arefinally hybridized with Cy3- and Cy5-labeled cDNA targets derived fromthe two biological samples being compared for differential geneexpression. After hybridization, the array is analyzed with afluorescence scanner and the relative amounts of an mRNA species in theoriginal two samples is defined as a ratio between the two fluorophoresat the homologous array element using specially designed software (Eisenand Brown, 1999; Hegde, et al., 2000; Schena, et al., 1995; Schena, etal., 1996; Wang, et al., 2001).

This useful technology, however, possesses recognized dataquality/reproducibility issues, that can limit its application tocomplex biological systems (Kerr and Churchill, 2001; Lee, et al.,2000). High experimental variability can arise through laboratorytechnical problems as well as normal biological variation (Pritchard, etal., 2001). Yue, et al., (2001), using Saccharomyces cerevisiae probesand complementary in vitro transcripts, demonstrated that the amount ofDNA bound to the glass slide is dependent, in part, on the concentrationof the DNA printed and that the amount retained by the slide is criticalfor good quality differential expression data (Yue, et al., 2001). Therange of detected values of known transcript ratios was compressed whenelements were printed at concentrations less than 100 ng/ul in water.Printing at more dilute printing concentrations exacerbated ratiocompression to the point where input transcript ratios of 30:1 or 1:30were detected as output ratios close to 1:1, illustrating that limitingbound probe results in an underestimation or failure to detectdifferential gene expression (Yue, et al., 2001). The concentration ofDNA printed, the printing buffer selected, and the glass coating willinfluence the amount of DNA retained by the slide after processing.Commonly used printing solutions include 3×SSC (saline sodium citrate),50% dimethyl sulfoxide (DMSO), and water (Eisen and Brown, 1999; Yue, etal., 2001). Diehl, et al., (2001) found that the addition of the PCRadditive betaine, which is known to normalize base pair stabilitydifferences, increase solution viscosity, and reduce evaporation rates,also greatly enhances probe binding to poly-L-lysine coated slides(Diehl, et al., 2001; Henke, et al., 1997; Rees, et al., 1993).Furthermore, probe saturation of the glass slide was obtained at a lowerprinting concentration of 250 ng/ul when betaine was present versus >500ng/ul in printing solutions without betaine, which can greatly increasethe number of potential slides produced from a single libraryamplification (Diehl, et al., 2001).

DESCRIPTION OF THE DRAWINGS

FIG. 1 is an evaluation of spotting solutions for post-blocking proberetention. Dilutions series fluorescein-labeled cDNA probe was printedin five different printing FIG. 1A: Fluorescein image immediately afterprinting. FIG. 1B: Fluorescein image (same array as panel A) afteraqueous post-processing. FIG. 1C: Plotted are the percent retentionvalues determined from the 100 ng/μl dilution element for each of the 3genes for the 6 printing solutions (n=35 elements, distributed over 35slides). Bar graphs ordered: GAPDH; B-actin, HBGR2. 3% DMSO 1.5M betainewas superior.

FIG. 2 indicates that the processed array fluorescein image isreflective of hybridized array performance. The experiment employs human10K probe cDNA arrays. FIG. 2A 1-A3 (nonaqueous blocking)/A4-6 (aqueousblocking): Array image immediately after printing (A1, A4), postprocessing (A2, A5), and homotypic hybridization with Cy5 and Cy3 directlabeled UACC903 RNA. FIG. 2B: Scatter plots of homotypic hybridizationson arrays processed with nonaqueous (top) and aqueous (bottom) methods.FIG. 2C: The variability in intensity Cy3/Cy5 ratio measurement (y-axis)is correlated with fluorescein signal to noise ratio (x-axis);nonaqueous (top) and aqueous (bottom) methods. Images were collectedusing same laser and PMT settings and are illustrated under the sameparameters using GenePix Pro Software. [Note: Loss of DNA afterprocessing step (A1 vs A2; A4 vs A5); white elements in panels A1 and A4are saturated]

FIG. 3 demonstrates that fluorescein signal to noise score (x-axis) of50 replicate pairs (100 slides) is predictive of correlation coefficientof Cy3/Cy5 ratio data between hybridized replicate arrays (y-axis). Allhybridizations are between Jurkat and UACC903 cDNA.

FIG. 4 demonstrates a tracking scheme for confirmation of plate orderand orientation from clone source plate to printed array usingfluorescein labeled probes. Panel A: Layout of asymmetric plate-specificnegative controls for first 4 clone source plates. Position Al of eachplate is removed to serve as an orientation marker; a second negativecontrol is used as a plate identifier. Panel B: 9600 element human cDNAarray printed on in-house-prepared poly-L-lysine coated slide using 16pins (set back). Subarrays generated by each pin are labeled. Subarray 1possesses position A1 from each source plate (A1 negative controlsgenerate the absence of 24/25 elements in the first (far left) column.Subarray 9 (enlarged) shows a correct series of negative controls forindicated plates; other probe plates are represented in other subarrays.Improper management of any plate at any point during array constructionwill disrupt this pattern. Note: observable pin clogging problem on pin2.

FIG. 5 shows a linear relationship between amount of labeled DNAdeposited on slide (x axis) and fluorescence detected (y axis). Toaccomplish this, multiple (n=4) serial dilutions in water (400 ng/ul to0.049 ng/ul) were generated from a pooled DNA sample derived from 384separate cDNA clone amplifications to account for different clone sizes(for example, single clones, one of 500 bp and one of 2000 bp, each at aconcentration of 150 ng/ul will have a molarity difference of 4-fold,and therefore a difference in fluorescence of 4-fold). Known volumespossessing known quantities (0.5 ul) of DNA were hand spotted on topoly-L-lysine slides, dried, and imaged. Fluorescein relativefluorescence units (RFU) were plotted against picograms of DNA (FIG. 2)to determine that, with the Packard ScanArray 5000 (laser power 70%; PMT80%), there are approximately 25 RFU detected per picogram DNA in thisexperiment. Average spot size in this experiment was >1500 microns indiameter with total DNA deposited being 50, 100, 200, 400, and 800 pgfor the points represented. Based upon a printing concentration of 150ng/ul, a probe deposition volume of 0.6 nl, and an 80% retention ratewith our new buffer, we estimate that approximately ˜75 pg of DNA isretained and available for hybridization. From these array elements,which measure ˜120 microns in diameter, we typically detect 10,000 RFUor 133 RFU per picogram, a discrepancy of approximately 5-fold. We knowthat fluorescein when in close proximity will self-quench, perhaps thisis why the detected fluorescence on mechanically generated spot is lessthan we would expect based on this experiment.

FIG. 6A demonstrates the use of fluorescein-labeled cDNA probes toevaluate spot/array morphology after printing and after fixing andblocking for in-house prepared versus commercially note differences spotmorphology and probe retention. Arrays 1-4 were printed on poly-L-lysinecoated slides produced at the Medical College of Wisconsin; ElectronMicroscopy Sciences, Fort Washington, Pa.; Polysciences Inc.,Warrington, Pa.; Cel-Associates, Pearland, Tex., respectively. Arrays5-13 were printed on aminosaline coated slides produced by AsperBiotech, Redwood City, Calif.; Apogent Discoveries, Waltham, Mass.;Bioslide Technologies, Walnut, Calif.; Erie Scientific, Portsmouth,N.H.; Genetix, St. James, N.Y.; Coming Inc, Corning N.Y. (GAPS); CorningInc, Corning N.Y. (GAPS II); Sigma, St. Louis, Mo.; TelechemInternational Inc, Sunnyvale, Calif., respectively. Arrays 14-15 wereprinted on epoxy coated slides produced by Telechem International Inc,Sunnyvale, Calif. (epoxy and super epoxy, respectively). FIG. 6Bdemonstrates competitive hybridization between Jurkat (Cy5) and UACC903(Cy3) labeled cDNA (30 ug total RNA labeled though incorporation of Cy5or Cy3-dUTP) hybridized to 10K human arrays printed on 16 differentcoated slides. Arrays 1-4 were printed on poly-L-lysine coated slidesproduced by MCW; Electron Microscopy Sciences, Fort Washington, Pa.;Polysciences Inc., Warrington, Pa.; Cel-Associates, Pearland, Tex.,respectively. Arrays 5-13 were printed on aminosaline coated slidesproduced by Asper Biotech, Redwood City, Calif.; Apogent Discoveries,Waltham, Mass.; Bioslide Technologies, Walnut, Calif.; Erie Scientific,Portsmouth, N.H.; Genetix, St. James, N.Y.; Corning Inc, Corning N.Y.(GAPS); Corning Inc, Corning N.Y. (GAPS II); Sigma, St. Louis, Mo.;Telechem International Inc, Sunnyvale, Calif., respectively. Arrays14-15 were printed on epoxy coated slides produced by TelechemInternational Inc, Sunnyvale, Calif. (epoxy and super epoxy,respectively).

FIG. 7 demonstrates imaging of a fluorescein-labeled oligonucleotide(70-mer) after printing (1A, 2A, 3A) and after fixing/blocking (2A, 2B,2C) in three different spotting solutions (A: 1.5M betaine/3% DMSO; B:3×SSC; C: 50% DMSO). Addition of a third color is useful for qualitycontrol of cDNA arrays as well as spotted oligonucleotide arrays.

SUMMARY OF THE INVENTION

In one embodiment, the invention is a method of directly visualizingprinted microarrays, comprising the steps of: (a) generating labeledprobes labeled with a first label, (b) constructing a microarray withthe labeled probes, wherein the microarray comprises a plurality ofprobe spots, and (c) examining the microarray to determine the amount ofprobe present at each probe spot. In one preferred form of theinvention, the labeled probes are either cDNA or oligonucleotides andthe first label is fluorescent. In another embodiment, the labeledprobes are proteins or antibodies.

In one embodiment, the labeled probes are labeled with a fluorescentprobe, such as fluorescein, and the examination of step (c) is via thedetection of relative fluorescence units and is by the use of a confocallaser scanner.

In one embodiment of the invention, the labeled DNA probes are between10 and 100,000 base pairs in length and the probes comprise 1fluorescent label molecule per DNA strand on average.

In another embodiment, the invention comprises the method describedabove additionally comprising the steps of (d) exposing the microarrayto labeled target molecules, wherein the labeled target molecules arelabeled with a second and third label, preferably a fluorescent label,and (e) examining the microarray to determine the amount of targethybridized to the probes.

In another embodiment, the invention is a microarray comprising (a) asurface and (b) labeled DNA probes attached to the surface in aplurality of spots, wherein each probe is labeled with a firstfluorescent label.

DESCRIPTION OF THE INVENTION

Controlling array fabrication variables is difficult because the arrayprinting is typically invisible until after hybridization. In thepresent invention, we have generated labeled probe arrays, as a means ofvisualizing element/array morphology and quantifying DNAdeposition/retention on the slide prior to hybridization. Directlabeling of probes separates slide coating, printing, and processingfrom hybridization and facilitates evaluation and optimization ofmethods. We have made the observation that slides coated, printed andprocessed together are not necessarily equivalents, and thatprehybridization imaging is predictive of hybridization performance.Therefore, prehybridization slide evaluation and selection can improvedata reproducibility and quality because slides that do not meet minimumstandards can be avoided.

A number of approaches have been described to address the problem ofdetermining DNA deposition/retention and array element morphology priorto experimental use of slides. It is possible to stain the fixed slideprior to hybridization with a DNA-binding fluorescent dye, such as SYBRGreen II or SYTO 61 (Battaglia, et al., 2000; Yue, et al., 2001).However, investigational use of the slide after quality control analysisrequires destaining, and potential changes in slide performance afterdestaining must be considered. The use of universal targets which willhybridize to every element of a microarray have also been reported (Yue,et al., 2001). While these hybridization-based techniques provideinformation as to the amount of DNA present within each element of thearray, they require sacrificing a slide from a batch of printed slidesfor quality control analysis and do not completely assure theinvestigator that the arrays actually used for experimentation areequivalent to those evaluated during quality control. Most recently,Ramakrishnan, et al., 2002 describe co-spotting a fluorescent dye withas a component of the printing buffer for monitoring mechanical aspectsof array fabrication. In this approach, the label is not covalentlyattached to the probe, and the spiked dye is presumably washed offduring blocking and fixing steps, so one does not know much probe wasretained on the array since the array is again invisible.

To circumvent this problem, we developed a means of directly visualizingprinted arrays by generating probes labeled with a fist label,preferably labeled with fluorescent-label primers such asfluorescein-labeled primers (excitation 488 nm/emission 508 nm), whichare spectrally compatible with the Cy5 and Cy3 dyes typically used fortarget labeling (Cy3 excitation 543 nm/emission 570 nm; Cy5 excitation633 nm/emission 670 nm) when using the GSI Luminonics ScanArray 5000confocal laser scanner. The narrow 10 nm bandwidth of this instrumentallows for excitation of Cy3 at 543 nm without co-excitation offluorescein, which would contaminate the Cy3 emission with its broademission tail. One might also wish to use luminescent or phosphorescentdyes. One may wish to use radioactive dyes. It is necessary that thefirst label be covalently coupled to the probe and that the first labelbe detectable and spectrally compatible. These probes are deposited,preferably as described below, in spots on a microarray surface,preferably a coated glass slide. By “spots”, we mean a deposit (or“printing”) of probes in discrete, specific area, such thathybridization of labeled targets to that specific area can be detected.

By “spectrally compatible” we mean that the trio of dyes are detectableand distinct from each other in a confocal laser scanner. Fluorescein,Cy5 and Cy3 are spectrally compatible using the GSI Luminonics ScanArray5000 confocal laser scanner. Other trios of dyes would be equallysuitable with this and other scanning systems. Other trios would includeany combination of fluorescein derivatives for lowest wavelength dye,including Alexafluor 488 (Molecular Probes, Eugene Oreg.). TheAlexafluor homologues for Cy3 and Cy5 could also be used for the middleand high wavelength dyes.

Our approach, which separates analysis of slide coating, printing, andprocessing from analysis of hybridization provides a method for 1) probeamplification control, 2) direct examination of array/elementmorphology, 3) determination of post-processed probe retention, and 4) ameans of bound probe quantitative quality control for improveddifferential gene expression analysis.

An advantage of this approach is the existence of a direct relationshipbetween detected relative fluorescence units (RFUs) and the amount ofDNA probe present on the slide, once unincorporated primer has beenremoved from the amplified probe, making DNA retention studies possible.

The present invention is a method and apparatus for performing amicroarray analysis. In one embodiment, the method comprises creating acDNA microarray wherein the cDNA is labeled with a first label,preferably a fluorescent label. Preferably, this first label isfluorescein. The first label must be spectrally compatible with secondand third labels. Target molecules are labeled with either the second orthird labels.

Microarrays can be fabricated using either amplified cDNAs as a sourceof probe material or, alternatively, a synthetic oligonucleotide.Oligonucleotide arrays, currently fall into two categories, those thatare fabricated through in situ synthesis, where the oligonucleotideprobe is synthesized directly on the array surface (example AffymetrixGeneChip, which uses 25-mers); or a spotted oligonucleotide array, wherethe fully synthsized oligo is spotted onto the array surface andattached through a variety of different chemstries (these oligos aretypically longer, i.e., 70-mers). The spotted oligo arrays offer theadvantage of being able to purify the probe that actually is attached tothe array (i.e., removal of short molecules that failed duringsynthesis), currently offer more flexibility in design, and can befabricated in the research laboratory. We envision that the presentinvention would encompass “spotted oligonucleotide” arrays. When werefer the microarrays comprising “oligonucleotides,” we are referring tocreation of full-length oligonucleotides that are then spotted onto thearray.

Since synthetic oligonucleotides are made in a 3′ to 5′ direction, theaddition of a compatible dye to the 5′-most position will result in thelabeling of only full-length molecules. A label of this nature would beuseful to spotted arrays since one could determine how much full-lengtholigonucleotide was present at each position on the array, as well asassess other array parameters, such as spot shape. In the case ofspotted oligq arrays, it would be possible to measure how probe wasredistributed over the array during the blocking steps, as we havedescribed for cDNA arrays.

It is possible to label proteins with dyes (including radioactive ones)for this same purposes. Therefore, the present invention comprisesprotein and antibody arrays. One would be able to confirm that theprotein is present, how much, shape of spot, and how well the proteincontained within the spot.

In one embodiment of the invention, one would examine the labeledmicroarray and directly measure the bound probe via detection of thefirst label. The Examples below describe preferable methods for thisanalysis. All the probes must be labeled. The cDNAs are typicallygenerated by PCR from plasmid clones. Labeling of this PCR product isaccomplished through the use of oligonucleotide primers that are 5′end-labeled with the first label. Since the primer becomes part of thePCR product, the cDNA is essentially covalently labeled once on each 5′end. Such primers for use in PCR sequencing, etc., are readily availablefrom oligonucleotide vendors. After analysis, one would be able todiscard microarrays that are that are not consistent a preset qualitycontrol standard. One might identify, in general, how much bound isnecessary to obtain highly reproducible results across high densityarrays. However, for key experiments, we are selecting arrays withsignal to noise ratios >0.90, average element fluoresceinintensity >3,000, and CV (coefficient of variation) of elementfluorescein intensity <10%.

In another embodiment of the present invention, one would expose themicroarray described above to the labeled targets and perform amicroarray binding analysis.

In another embodiment of the present invention, a microarray is providedwherein the probe is labeled with a first label. Preferably this labelis fluorescent and the array is either a cDNA or an oligonucleotidearray. In another embodiment of the present invention, the array is aprotein array or an antibody array.

The array of the present invention is preferably created by thefollowing steps: The cDNA array is typically prepared by firstamplifying by PCR the cDNA clone inserts from their plasmid vectors.This can be done in a 96-well format or a 384-well format. We use384-format for PCR and all subsequent steps. Clones that serve as asource of cDNA templates can be commercial vendor, such as ResearchGenetics or the I.M.A.G.E. Consortium, or personal cDNA libraries. PCRreactions to amplify these cDNA clone inserts can be conducted directlyfrom bacterial culture or from purified plasmid template. In eithercase, the oligonucleotide primers are labeled with a first fluorescentlabel. We have selected fluorescein due to our instrumentation and itscompatibility with Cy3 and Cy5 on our instrumentation. After PCR of the20,000-plus clones to be printed on the chip, the PCR reactions must bepurified. This is done for a number of reasons, including removing PCRreaction components and buffer. We have chosen a size exclusionfiltration approach since it removes most of the unincorporated labeledoligonucleotide. After purification, the 384 plate is quantified, drieddown, and reconstituted in 1.5M betaine/3%DMSO for printing. Probematerial is then printed onto coated glass slides as “spots”. Since thePCR product has been purified, and unincorporated labeled primers areremoved, the measured fluorescence on the array is proportional to theamount of PCR product present on the slide versus due to PCR productplus primer. This approach is different than other visualization methodsbecause the probe is covalently attached to the label, versus a staininginteraction or hybridization. This method allows every slide to have QCanalysis before use.

The preferred first label is fluorescein or a fluorescein derivative.Fluorescein derivatives have been the most commonly used label forbiological molecules. In addition to its relatively high absorptionproperties, excellent fluorescence quantum yield and good watersolubility, fluorescein has an excitation maximum (494 nm) that closelymatches the 488 nm spectral line of the argon-ion laser, making it auseful fluorophore for confocal laser-scanning microscopy applications.Our selection of fluorescein as the “first label” was first driven byfact that it is compatible with Cy3 and C5 when using the ScanArray5000, and second by the fact that this fluorophore is relativelyinexpensive and readily available as a 5′ end-label on oligonucleotideprimers.

Unfortunately, many confocal laser scanners do not possess theperformance specifications to support the use of a three-color system aswe describe here using fluorescein. In our system, the followingexcitation/emission wavelengths are used: Fluorescein 488 nm/508 nm; Cy3543 nm/570 nm; Cy5 633 nm/670 nm. The key feature of the Scan Array 5000instrument that makes 3 dyes possible, besides the fact that it has therequired laser to excite fluorescein at 488 nm, is the fact that it canexcite and read these wavelengths with a very narrow bandwidth (±5 nm).Practically, this means that Cy3 can be excited without co-excitingfluorescein; since fluorescein has such a broad emission spectrum, if itwere to be excited when trying to excite Cy3, the Cy3 emission spectrumwould be contaminated. This situation is likely to change as both thefluorescent labels and instrumentation continue to improve, allowingmore flexibility in dye and instrument selection in three-colorapplications. None the less, the strategy as described in this reportperforms well. We are confident fluorescent labeling of the probes doesnot interfere with the subsequent detection of second and third label(Cy3 and C5) hybrids, because (1) scanning of slides prior tohybridization shows no signal for either the second or third label (Cy3or Cy5 in our Example); and (2) second/third label (Cy3/Cy5) scatterplots pass through the origin with no evidence of the detected second orthird label (Cy3 or Cy5) signal being negatively influenced by aquenching effect nor positively influenced by carryover signal.Furthermore, all of our arrays (including those shown in FIGS. 2, 4, 6Aand 6B) possess a series of fluorescein-labeled Arabidopsis thalianaprobes to be used as positive (in combination with homologous in vitrotranscript) and negative controls. These probes generate no signal undersecond or third label (Cy3 or Cy5 in our Example) scanning conditionseither before or after hybridization in the absence of labeled in vitrotranscript.

Direct measurement of the bound probe available for hybridization hasother important advantages. Electrophoretic analysis of probeamplification efficiency can be greatly reduced since failed PCRs can beidentified and recorded through analysis of fluorescein signalintensity. Precious clinical target material can be conserved throughreduction of replicates necessary because poor quality slides can beavoided. Quality-based prehybridization selection results in a higherprobability of successful experiments and reduced overall cost.Preferably, we select arrays with signal to noise ratios >0.90, averageelement fluorescein intensity >3,000, and CV (coefficient of variation)of element fluorescein intensity <10%.

In one version of the present invention, one would introduce targetslabeled with second and third labels. In a preferred embodiment, themethod would comprise the following steps: RNA samples are isolated fromthe tissues that are being compared for gene expression. Labeled cDNAtargets are derived from these samples by reverse transcription, wherebyCy 3 is incorporated into one sample and Cy5 is incorporated into theother. Equal amounts of the two labeled samples are hybridized to thearray, allowing the labeled targets to base pair with their respectivehomologous probe on the array. The array is the washed and scanned forboth wavelengths in a confocal laser scanner and the images analyzed bysoftware. Transcripts in both samples in equal amounts will give rise todye ratios of “1”; whereas transcripts over or under expressed relativeto the other sample will give rise to ratios deviating from one.

EXAMPLES Example 1 Three Color cDNA Microarrays: Quantitative Assessmentthrough the use of Fluorescein Labeled Probes

Results:

Human probes for glyceraldehyde 3-phosphate deydrogenase-1 (GAPDH),B-actin, and glutamate receptor-2 (HBGR2) (IMAGE Consortium 50117,34357, and 43622, respectively) were serially diluted and printed in 50%DMSO, 3×SSC, water, 1.5M betaine, 1.5M betaine/3×SSC (Diehl, et al.,2001) and 1.5M betaine/3.1% DMSO. Arrays were evaluated for spotmorphology (size/shape) and DNA retention was measured by scanningarrays immediately after printing and again after post-processing. Only30% of probe is retained by poly-L-lysine coated glass slides afterpost-processing when the commonly used printing solutions water, 50%DMSO, or 3×SSC are used [FIGS. 1A and 1B]. Probes printed with 50% DMSOresulted in 151.1±5.9 micron diameter array elements compared to120.6±5.4 micron diameter elements for those printed in water or 3×SSC(with or without 1.5M betaine), therefore, DMSO was titrated in aneffort to control spot size. The use of 3% DMSO/1.5M betaine resulted inthe highest average probe retention on the slide (>70%), more than twicewhat is observed with commonly used printing solutions, as well asoptimal average spot size (<130 microns) [FIG. 1C]. Preparation of DNAprobe is the most time consuming and expensive component of high-densityarray construction and making efficient use of prepared probe throughhigh retention an important ongoing issue.

The critical post-arraying blocking process, where unreacted primaryamines are converted to carboxylic moieties, is typically performed withsuccinic anhydride in an aqueous borate buffered1-methyl-2-pyrrolidinone (Dolan, et al., 2001; Eisen and Brown, 1999;Schena, et al., 1995; Schena, et al., 1996). Generation offluorescein-labeled arrays enabled direct hybridization-free comparisonof this traditional blocking process to blocking with succinic anhydridein the non-polar, non-aqueous solvent 1,2-dichloroethane (Diehl, et al.,2001). Processing with the nonaqueous method resulted in arrays withvery low background fluorescein signal levels compared to the aqueousblocking method [FIG. 2A 2 versus 2A5] where background levels increasedas a function of printed DNA concentration (data not shown). Theprehybridization image quality was predictive of slide performance inhomotypic hybridizations employing UACC903 RNA where arrays processedwith the nonaqueous method generated images with higher overall signalintensity and fewer outliers [2A3 versus 2A6, 2B].

Image quality was assessed with Matarray software (Wang, et al., 2001),which employs a spatial and intensity dependent algorithm for spotdetection and signal segmentation. Matarray also generates a compositequality score (q_(com)) that is defined for each spot on the arrayaccording to size, signal-to-noise value (signal/signal+noise),background uniformity and saturation status (Wang, et al., 2001).Variation in Cy5/Cy3 intensity ratio values correlated with thefluorescein q_(com) score and revealed an overall lower spot qualitywith the nonaqueous method that impacts data quality [FIG. 2C]. Usingsimultaneously produced 10,000 probe arrays, mean signal to noisequality score (signal/[signal+noise]) per element of 0.93±0.04 (n=15)were observed with the non-aqueous method versus 0.71±0.02 (n=15) withthe aqueous method. Probe signal measurements of 6-9 fold over noisewere observed on arrays processed with the nonaqueous blocking methodand values slightly less for those arrays aqueously processed; thesevalues are sufficient for credible measurement of bound probe. Theseobservations are consistent with the notion that aqueous blockingmethods result in partial re-dissolving and re-deposition of printedDNA, generating higher background.

Slides that are coated, printed, and processed together do notnecessarily result in equivalent arrays. One hundred slides eachpossessing a 10,000 human probe array were simultaneously printed,nonaqueously processed, and evaluated. The average fluoresceinsignal/slide varied between processed slides from 4,500 RFU to 20,000RFU (10,770±4,202); while overall slide signal to noise values rangedfrom 0.85 to 0.95 (mean=0.92±0.03). Competitive hybridizations betweenUACC903 and Jurkat cDNA on arrays, selected from three independentprintings of the same probe set, with high DNA/element and lowbackground values were compared to those performed on arrays with lowDNA/element and/or high background values. When comparing hybridizationresults between replicate pairs of differing quality (n=50 pairs), adirect and significant relationship (R²=0.80, p<0.001) was observedbetween prehybridization fluorescein image quality and replicateconsistency, illustrating that microarray data quality can be improvedthrough prehybridization slide selection based upon quality analysis.The observation of a relationship between pre and post hybridizedimage/data quality is completely consistent with our previous report inthat prehybridized arrays possessing low signal to noise scores giverise to hybridized arrays with low signal to noise scores andhybridization data from such arrays do not correlate well with eachother (Wang, et al., 2001). Selection of quality arrays does notnecessarily guarantee high replicate Cy5/Cy3 ratio correlation, becauseRNA samples, target labeling, hybridization, washing, laboratorytechnique, and image collection are sources of variation, as indicatedby the three outliers observed in FIG. 3. It must be emphasized that the100 hybridizations represented in FIG. 3 were performed by multiplelaboratory personnel utilizing multiple labeling reactions of the sameRNA.

Methods:

The Research Genetics (Huntsville, Ala.) sequence-verified humanlibrary, consisting of 41,472 clones was used as a source of probe DNA.The library was reformatted from 96 to 384-format and subsequentlymanipulated using 0.5 μl and 5 μl volume 96 and 384 slot pin replicatortools (VP Scientific, San Diego, Calif.). Clone inserts were directlyamplified in 384-well format from 0.5 μl bacterial culture using 0.26 μMof each vector primer [array F:5′-fluorescein-CTGCMGGCGAT-(fluorescein)TAAGTTGGGTMC-3′ (SEQ ID NO:1)and array R:5′-fluorescein-GTGAGCGGAT-(fluorescein)MCAAMTTTCACACAGGAACAGC-3′ (SEQ IDNO:2)] (Integrated DNA Technologies, Coralville, Iowa) in a 20 μlreaction consisting of 10 mM Tris-HCl pH8.3, 3.0 mM MgCl₂, 50 mM KCl,0.2 mM each dNTP (Amersham, Piscataway, N.J.), 1M betaine, and 0.25 UTaq polymerase (Roche, Indianapolis Ind.). Reactions were incubated at95° C. for 5 minutes and 35 cycles of 95° C. for 1 minute, 55° C. for 1minute, 72° C. for 1 minute, and terminated with a 7 minutes hold at72°. PCR products were routinely analyzed for quality by 1% agarose gelelectrophoresis analysis. Products were purified by size exclusionfiltration using the Multiscreen 384 PCR filter plates (Millipore,Bedford, Mass.) to remove unincorporated primer and PCR reactioncomponents. Forty wells of each 384-well probe plate were quantified bythe PicoGreen assay (Molecular Probes, Eugene, Oreg.) according to themanufacturers instructions, dried down, and reconstituted at 125 ng/μlin 3% DMSO/1.5M betaine.

Microarrays possessing a density of 10,000 probes/slide were printedonto poly-L-lysine slides using a GeneMachines Omni Grid printer (SanCarlos, Calif.) with 8 Telechem International SMP3 pins (Sunnyvale,Calif.). Slides were post-processed using the previously describedaqueous (Eisen and Brown, 1999) or nonaqueous (Diehl, et al., 2001)protocols. Slide coating, isolation of mRNA, labeling, and hybridizationwere performed as described previously in Hedge, et al., 2000; Schena,et al., 1995; and Yue, et al., 2001. After hybridization, arrays werescanned with a ScanArray 5000 (GSI Luminonics, Billerica, Mass.) andimage files were obtained. Array image files were analyzed with theMatarray software (Wang, et al., 2001).

Example 2 Use of a Three-Color cDNA Array Platform to Measure andControl Available Bound Probe for Improved Data Quality andReproducibility

We directly evaluated the impact of differing amounts of bound probe onhybridized replicate data correlation, and investigated the performanceof 15 different vendor-supplied coated slides in terms of DNA retentionand hybridization performance. Furthermore, utilizing our three-colorcDNA microarray platform, we developed and describe here a novel probetracking system for ascertainment of proper plate order and orientationfrom culture growth, amplification, and purification, through printingof probes onto the array.

Materials and Methods:

Library Growth and Tracking

The Research Genetics (Huntsville, Ala.) sequence-verified humanlibrary, consisting of 41,472 clones was used as a source of probe DNA.The library was reformatted from 96 to 384-format and subsequentlymanipulated using 0.5 μl and 5 μl volume 96 and 384 slot pin replicatortools (VP Scientific, San Diego, Calif.). Cultures were grown in 150 ulTerrific Broth (Sigma, St. Louis, Mo.) supplemented with 100 mg/mlampicillin in 384 deep-well plates (Matrix Technologies, Hudson, N.H.)sealed with air pore tape sheets (Qiagen, Valencia, Calif.) andincubated with shaking for 16-18 hours. A unique asymmetric pattern oftwo negative controls per 384 culture plate was created by transferringthe contents of the selected wells to a new 384 plate and updating theclone tracking database accordingly. The plate-specific negative controlpattern was created by removing position A1 (to establish an orientationmarker) and one additional plate-specific wellA2 (FIG. 4).

Clone inserts were amplified in duplicate in 384-well format from 0.5 ulbacterial culture diluted 1:8 in sterile distilled water or from 0.5 ulpurified plasmid (controls only) using 0.26 μM of each vector primer{SK865 5′-fluorescein-GTC CGT ATG TTG TGT GGA A-3′ (SEQ ID NO:3) andSK536: 5′-fluorescein-GCG AAA GGG GGA TGT GCT G-3′ (SEQ ID NO:4) (Yue,et al., 2001)} (Integrated DNA Technologies, Coralville, Iowa) in a 20μl reaction consisting of 10 mM Tris-HCl pH 8.3, 3.0 mM MgCl₂, 50 mMKCl, 0.2 mM each dNTP (Amersham, Piscataway, N.J.), 1M betaine (Henke,et al., 1997; Rees, et al., 1993) and 0.50 U Taq polymerase (Roche,Indianapolis Ind.). Reactions were amplified with a touchdown thermalprofile consisting of 94° C. for 5 minutes; 20 cycles of 94° C. for 1minute, 60° C. for 1 minute (minus 0.5° per cycle), 72° C. for 1 minute;and 15 cycles of 94° C. for 5 minutes; 20 cycles 94° C. for 1 minute,55° C. for 1 minute, 72° C. for 1 minute; terminated with a 7 minuteshold at 72° (Don, et al., 1991; Hecker and Roux, 1996; Roux and Hecker,1997). PCR products were routinely analyzed for quality by 1% agarosegel electrophoresis analysis. Products from replicate plates pooled andthen purified by size exclusion filtration using the Multiscreen 384 PCRfilter plates (Millipore, Bedford, Mass.) to remove unincorporatedprimer and PCR reaction components. Forty wells of each 384-well probeplate were quantified by the PicoGreen assay (Molecular Probes, Eugene,Oreg.) according to the manufacturers instructions; alternatively, 1 ulof each 384 plate well was pooled and absorbance at 260 nm read directlyfor quantification. After quantification, all plates were dried down,and reconstituted at 125 ng/μl in 3% DMSO/1.5M betaine.

Poly-L-lysine coated slides were prepared in-house as previouslydescribed (Eisen and Brown, 1999). Nine different commercially availableaminosaline coated slides (Apogent Discoveries, Waltham, Mass.; AsperBiotech, Redwood City, Calif.; Bioslide Technologies, Walnut, Calif.;Corning Inc, Corning N.Y.; Erie Scientific, Portsmouth, N.H.; Genetix,St. James, N.Y.; Sigma, St. Louis, Mo.; Telechem International Inc,Sunnyvale, Calif.) and 3 different commercially available poly-L-lysinecoated slides (Cel-Associates, Pearland, Tex.; Electron MicroscopySciences, Fort Washington, Pa.; Polysciences Inc., Warrington, Pa.) wereobtained for evaluation. Lastly, two types of epoxy-coated slide(Telechem International Inc, Sunnyvale, Calif.), and slides coated witha proprietary chemistry obtained from Full Moon Biosystems (Sunnyvale,Calif.) were obtained. In all 16 different slide sources, includingpoly-L-lysine slides prepared in-house, belonging to 3 generalcategories, were evaluated in terms of spot morphology and DNAretention.

Microarrays possessing a density of 9,600 human probes/slide wereprinted onto coated slides using a GeneMachines Omni Grid printer (SanCarlos, Calif.) with 16 Telechem International SMP3 pins (Sunnyvale,Calif.) at 40% humidity and 22° C. (72° F.). To control pin contactforce and duration, the instrument was set with the following Z motionparameters, velocity: 7 cm/sec, acceleration: 100 cm/sec², deceleration:100 cm/sec².

Slides were post-processed using the previously described nonaqueousprotocol (Diehl, et al., 2001). Slide coating, isolation of mRNA,labeling, and hybridization were performed as described previously inHedge, et al., 2000; Schena, et al., 1995; and Yue, et al., 2001. Imagefiles on all arrays were collected after printing (fluorescein), afterblocking (fluorescein), and again after hybridization (Cy3 and Cy5) witha ScanArray 5000 (GSI Luminonics, Billerica, Mass.). Array image fileswere analyzed with the Matarray software (Wang, et al., 2001).

Results and Discussion. Quality array construction requires generationof adequate amounts concentrated probe and printing probes in a knownordered fashion onto coated glass slides. We have opted to reformatlibraries from 96 to 384-format for culture growth/archiving, PCR,purification, and printing. This has reduced the number of plates of our41,472 human clone library from 432 to a more manageable 108. A highlyoptimized touchdown PCR protocol has been developed whereby 1-2 ugpurified probe material is recovered from 2 pooled and purified 20 ulPCR reactions. Duplicate reactions compensate for random PCR failures,enabling overall PCR success rates, based upon gel analysis, of ˜90%.Recovery of >1 ug purified probe enables printing >2000 arrays peramplification (assuming: 4 ul plate dead volume, printing at 150 ng/ul,and 250 nl/pickup/100 slides using the TeleChem SMP3 pins). The factthat the array is visible prior to hybridization allows for spots thatare not present on the array due to PCR failure or mechanical problems(clogged pin) to be tracked, eliminating a potential source oferror/variance between replicate slides. This has lead to thedevelopment of a tracking system, which utilizes a unique pattern ofnegative controls for each clone source plate enabling a means to assessthat all plates have had order and orientation maintained from the clonesource plate through growth, PCR, pooling, purification, and finallyprinting (FIG. 4).

A number of critical parameters, including DNA concentration, printingbuffer, slide surface, temperature, humidity, and print head velocitycan influence the amount of DNA deposited, retained, and ultimatelyavailable for hybridization on the slides surface (Diehl, et al., 2001;Yue, et al., 2001; Hegde, et al., 2000). Previously, we evaluated theretention characteristics of 50% DMSO, 3×SSC, water, 1.5M betaine, 1.5Mbetaine/3×SSC and 1.5M betaine/3.1% DMSO on poly-L-lysine coated slidesprepared in our own laboratory and found that on this surface, 1.5Mbetaine/3% DMSO offered the best retention (˜70%) under the conditionsdescribed in the Methods section. Since printing of labeled probesenables direct measurement of DNA deposition and retention, we evaluated15 different commercially available coated slides, in an attempt toidentify surfaces that offered the best performance in terms ofbackground fluorescence, spot morphology, amount of DNA ultimatelyavailable for hybridization, and competitive hybridization performanceusing Cy3 and Cy5 labeled Jurkat and UACC903 cDNA. Including ourin-house prepared slides, 18 different prepared surfaces were availablefor comparison: poly-L-lysine (n=4), aminosaline (n=9), epoxy (n=2), anda single unknown proprietory chemistry (Full Moon Biosystems; Sunnyvale,Calif.). A single 9600 element human cDNA array was spotted onto eachslide in 1.5M betaine/3% DMSO; additionally, a 384 plate of human cDNAprobes in water, 3×SSC, and 50% DMSO were spotted onto each slide inorder to control for the possibility that some of the commercialsurfaces may have been optimized for spotting with these more commonlyused solutions. Five replicate arrays for each slide type weregenerated. These five replicates were evenly distributed over thearrayer deck (capacity 100 slides) by arranging the slides into 5 groupsof 18 to account for any variance introduced by placement in the printorder (ie first versus last). Prior to printing, background Cy3, Cy5,and fluorescein fluorescence was measured. Fluorescein background wasobserved on all poly-L-lysine slides except for those produced in-house.Fluorescein background was also observed on 6 of aminosaline slides(Asper Biotech, Corning, Erie Scientific, Genetix, Telechem), as well ason the proprietary surface from Full Moon Biosciences. Cy3 backgroundwas again observed on all 3 commercial poly-L-lysine slides but notthose prepared in-house. No Cy3 background was observed on any of theaminosaline or epoxy slides. Slight Cy5 background was observed on only2 commercial poly-L-lysine slides (Electron Microscopy Sciences,Polysciences Inc.).

Fluorescein images were obtained immediately after printing and againafter post-processing to measure DNA deposited and retained. Thisrequired a confocal laser scanner calibration method; to ensureconsistent image collection, therefore we set the laser voltage power onthe instrument (typically ˜70%) against the FluorIS (CLONDIAG, Jena,Germany), a non-bleaching, reusable, calibration/standardization toolfor fluorescein, Cy5, and Cy3 image collection, while holding the photomultiplier tube (PMT) parameters constant (80%). Under these conditions,multiple scans of the same array are possible with little to nodetectable fluorescein signal degradation.

PCR products amplified from cDNA clones using single-labeledoligonucleotide primers possess two dyes per double-stranded product andproduct sizes typically range from ˜500 bp to ˜2000 bp. Therefore, it ispossible to mathematically predict the amount of fluorescence generatedper picogram of amplified and purified PCR product. However, a directmeasurement avoids the error introduced through variables such asfluorescein-fluorescein proximity quenching effects. To accomplish this,multiple (n=4) serial dilutions in water (x ng/ul to y ng/ul) weregenerated from a pooled DNA sample derived from 384 separate cDNA cloneamplifications to account for different clone sizes. Known volumespossessing known quantities of DNA were spotted on to poly-L-lysineslides, dried, and imaged. Fluorescein relative fluorescence units (RFU)were plotted against picograms of DNA (FIG. 5) to determine that, withthe Packard ScanArray 5000 (laser power 70%; PMT 80%), there areapproximately Z picograms/RFU.

Illustrated in FIG. 6 are images of human cDNA arrays possessing 9600elements spotted on the 16 different coated surfaces using 10% DMSO/1.5Mbetaine as a printing buffer. Images of arrays immediately afterprinting (FIG. 6A), after processing (FIG. 6B), and after competitivehybridization to labeled Jurkat and UACC903 cDNA (FIG. 6C) are shown.All hybridizations were prepared from a single pool of labeled cDNAs tonormalize any variances introduced through individual reversetranscription reactions. This experiment illustrates that not all vendorsupplied coated slides are, equivalent and probe labeling can be used tomeasure the amount of material available on the array surface.

To further evaluate the impact of the amount of bound probe available onthe overall quality of gene expression data obtained from cDNAmicroarrays, two hundred 9600 element human cDNA probes were printedonto 100 slides with a single pin loading per probe. This resulted in aseries of arrays with an average bound probe per element available forhybridization ranging from X pg/element to Y pg/element. The overallgoal of this experiment was to establish a general guideline as to howmuch DNA is needed per element to ensure that probe is in excessrelative to labeled target for the majority of transcripts one mayencounter in a standard microarray experiment. This would enable thefuture identification of those arrays possessing insufficient boundprobe, which as replicates would introduce experimental variability.This series of arrays was hybridized again to a pool of labeled Jurkatand UACC903 cDNAs to normalize any differences between individual targetlabeling reactions.

References:

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1. A method of directly visualizing microarrays, comprising the stepsof: a) generating labeled probes labeled with a first label, b)constructing a microarray with the labeled probes, wherein themicroarray comprises a plurality of probe spots, and c) examining themicroarray to determine the amount of probe present at each probe spot.2. The method of claim 1 wherein the probes are DNA molecules.
 3. Themethod of claim 1 wherein the probes are selected from the groupconsisting of cDNA and oligonucleotides.
 4. The method of claim 1wherein the probe is selected from the group consisting of proteins andantibodies.
 5. The method of claim 1 wherein the labeled probes areattached to the microarray surface via electrostatic and covalent bonds.6. The method of claim 1 wherein the first label is fluorescent.
 7. Themethod of claim 1 wherein the labeled probes are labeled withfluorescein.
 8. The method of claim 1 wherein the label is selected fromthe group consisting of fluorescent, radioactive, phosphorescent andluminescent labels.
 9. The method of claim 5 wherein the examination ofstep (c) is via the detection of relative fluorescence units and is bythe use of a confocal laser scanner.
 10. The method of claim 1 wherein apreferred amount of probe has been determined and the microarrays areevaluated using this preset amount.
 11. The method of claim 5 whereinthe fluorescently labeled probes of step (a) are generated via labeledprimers.
 12. The method of claim 2 wherein the labeled probes arebetween 10 and 100,000 base pairs in length.
 13. The method of claim 2wherein the probes comprise 1 label molecules per DNA strand on average.14. The method of claim 1 additionally comprising the step of (d)exposing the microarray to labeled target molecules wherein the labeledtarget molecules are labeled with a second and third label.
 15. Themethod of claim 14 comprising the additional step of (e) examining themicroarray to determine the amount of target bound to the probes. 16.The method of claim 1 wherein the microarray comprises apoly-lysine-coated glass slide.
 17. The method of claim 2 whereinDMSO/1.5 M betaine is used during the attachment of the probes to themicroarray.
 18. The method of claim 1 wherein step (c) comprisesmeasurement of image quality as assessed by software which employs aspatial and intensity-dependent algorithm for spot detection and signalsegmentation.
 19. The method of claim 1 wherein the microarrays possessa density of 3,000-10,000 probes/slide.
 20. A printed microarraycomprising a) a surface, and b) labeled probes attached to the surfacein a plurality of spots, wherein each probe is labeled with a firstlabel, wherein the probe is selected from the group consisting ofspotted oligonucleotides, cDNA, protein and antibodies.
 21. Themicroarray of claim 20 wherein the probe is DNA.
 22. The microarray ofclaim 20 wherein the probe is selected from the group consisting ofnucleic acids, protein, and antibodies.
 23. The array of claim 20wherein the surface is a glass slide.
 24. The array of claim 20 whereinthe surface is coated with a coating selected from the group consistingof poly-L-lysine, aminosaline, epoxy, and aminoallyl.
 25. The array ofclaim 20 wherein the first label is fluorescent.
 26. The array of claim25 wherein the first fluorescent label is fluorescein.
 27. The array ofclaim 20 wherein the first label is selected from the group consistingof fluorescent, luminescent, radioactive or phosphorescent labels.